Methods for size fractionating mixtures of single-strand or double-strand (duplex) nucleic acids are crucial to a variety of analytical and preparative techniques in biochemistry. One important example is restriction analysis, in which duplex DNA is digested with selected restriction enzyme(s), fractionated according to digest fragment size, then analyzed for fragment positions. This method is widely used in molecular cloning to determine the number and arrangement of restriction sites in a cloning vector, and to confirm insert location and/or orientation in the vector.
Restriction analysis is an important tool for genetic mapping as well, since it allows relatively long pieces of chromosomal DNA to be mapped, and ultimately sequenced, on the basis of restriction fragment sizes and overlap. In a related application, the discovery of linkages between a number of genetic diseases and restriction fragment length polymorphisms in humans has provided a tool for screening individuals for these genetic diseases.
Rapid DNA sequencing methods currently in use also rely on the ability to fractionate DNA fragments--typically single-strand fragments--on the basis of size. Both enzymatic sequencing techniques, in which random-termination fragments are generated enzymatically in the presence of dideoxynucleotides (Sanger), and chemical methods in which random-termination fragments are generated chemically (Maxam), rely on fractionation and discrimination of the fragments on the basis of fragment size. In particular, the fractionation method must be capable of distinguishing fragments which differ from each other by one nucleotide only.
In addition, size fractionation of nucleic acid fragments is valuable for isolating and purifying DNA or RNA fragments. In molecular cloning, it is common to fractionate restriction fragments to obtain selected fragments for vector construction. In oligonucleotide synthesis, it is generally desirable to purify fragments having the desired oligonucleotide sequence and subunit number.
Heretofore, standard methods available for size-fractionating nucleic acids fragments have used a solid or semi-solid gel matrix for electrophoretic fragment separation. In the case of larger molecular weight fragments, typically greater than about 1,000 bases, the preferred gel material is agarose, where the concentration of the agarose may vary from about 0.3%, for separating fragments in the 5-60 kilobase size range, up to about 2%, for separating fragments in the 100-3,000 basepair range (Maniatis). Smaller size fragments, typically less than about 1,000 basepairs, are usually separated in polyacrylamide gel. The concentration of acrylamide polymer can range from about 3.5%, for separating fragments in the 100-1,000 basepair range, up to about 20%, for achieving separation in the size range 10-100 basepairs.
More recently, DNA fragment separation by capillary electrophoresis (CE) has been proposed (Cohen, 1987, 1988, Compton, Kaspar). In one approach (Kaspar), fragments are separated in a gelled polyacrylamide medium within the tube. This approach shares many of the limitations of conventional acrylamide or agarose electrophoresis: the inconvenience of handling a polymerized gel, relatively long run times, and narrow size distribution of fragments which any given concentration of gel is capable of resolving.
Alternatively, it has been proposed to carry out fragment separation by CE in a buffer solution, without any separation medium (Cohen, 1987, 1988, and Compton). Generally, this approach has not produced consistent or easily interpretable results.